Apparatus for detecting microbial pathogens employing a cushioning agent

ABSTRACT

A method and apparatus are disclosed which provide for improved rapid quantitative analysis of a blood sample for the presence of microbial pathogens. The blood sample is lysed and deposited on a high density, water immiscible, hydrophobic, nontoxic, liquid cushioning agent and subjected to centrifugation. Microbial pathogens contained in the lysed blood sample will collect in a layer adjacent the interface of the cushioning agent and the blood sample residue and, in this concentrated form, can easily be separated from the residual portion of the blood sample for culturing and quantitative counting.

This is a division of application Ser. No. 739,274 filed Nov. 5, 1976now U.S. Pat. No. 4,131,512.

BACKGROUND OF THE INVENTION

This invention relates to a novel method and means for detectingmicrobial pathogens. In another aspect, this invention relates to anovel technique for selectively separating microorganisms from a samplefluid. In still another aspect, this invention relates to a method ofseparating microbial pathogens from a lysed blood sample which maycontain other components, such as antimicrobial constituents of bloodand medicants present in the blood sample without the use of specializedsolid or liquid filter media. A further aspect of this invention relatesto an apparatus for use in the detection of microbial pathogens whichprovides improved recovery of microorganisms. In still another aspect,this invention relates to a novel method and apparatus for diagnosingsepticemia.

Septicemia, which is the presence of pathogenic microorganisms in theblood, is one of the most serious types of infections encountered. Eventhough modern medicine has provided an armament of antibiotics andfungal drugs, the mortality rate from septicemia is approximately 25percent. In addition, when shock accompanies septicemia, the mortalityrate increases to over 60 percent. Debilitating diseases, major surgery,administration of immuno suppressive drugs or anti-cancer medicationcause patients to be particularly prone to septicemia.

Early administration of appropriate antibiotic therapy is important infighting septicemia. Consequently, it is imperative that the physicianknow as rapidly as possible, not only whether the patient hassepticemia, but also the identity of the affecting microorganisms andthe susceptibility of the microorganisms to antibiotic agents. Thus,proper and timely diagnosis of septicemia depends upon very rapid andefficient quantitative analysis of the patient's blood. Further, it isimperative during the quantitative analysis of the patient's blood thatthe blood sample not be contaminated with pathogens from the laboratoryenvironment.

Three analytical systems have been conventionally utilized to determinethe presence of microorganisms in a body fluid. These conventionalsystems include the liquid broth culture technique, the so-called pourplate method and the filtration method using a solid matrix filter. Eachof these systems has its drawbacks, and none of the systems provide forrapid detection of microorganisms in the blood sample. Generally, theliquid broth method is not quantitative, and the pour plate method andfiltration method (using a solid matrix filter) are open systems subjectto external contamination, e.g., the introduction of pathogens into theculture by the laboratory atmosphere and personnel.

Recently, an improved method and apparatus has been developed fordetermining the presence of microbial organisms within a sample fluidincluding, for example, blood. This method is disclosed in U.S. Pat. No.3,928,139, issued Dec. 23, 1975 and entitled "Detection of MicrobialPathogens". According to this improved method, rapid and quantitativedetection of microbial pathogens from a sample of body fluid is providedby employing a liquid filter medium. The sample fluid is placed on theliquid filter medium within a confined sterile zone. The liquid filtermedium has a density greater than the sample fluid and comprises asterile aqueous solution which will selectively receive microbialpathogens from the sample fluid. The confined sterile zone is thereaftersubjected to centrifugation to force the sample fluid against the liquidfilter medium and cause microbial pathogens to selectively pass thereinand thereby separate from the mass of the body fluid sample. Next, theliquid filter medium containing the microbial pathogens is separatedfrom the remainder of the sample fluid and portions of the liquid filtermedium are subjected to various culturing conditions.

The improved method described above does provide a very rapid andefficient procedure for separating microbial pathogens from a samplefluid. According to the preferred embodiment of the liquid filter mediummethod, the blood sample is lysed prior to the centrifugation step whichcauses the microbial pathogens to be selectively received by the liquidfilter medium. Other pretreating agents, such as anti-coagulating agentsare also used to prepare the blood sample. Some ingredients of thepreferred liquid filter media employed by the improved method discussedabove are incompatible with some of the pre-treating and/or lysingagents. Furthermore such agents will admix with the liquid filter mediumif added thereto prior to the time that the blood sample is added to theconfined sterile zone, and once so admixed such agents cannot defusefrom the liquid filter medium rapidly enough to effectively treat theblood. Therefore, it is necessary either to subject the blood samples tothe possibility of external contamination by admixing the blood samplewith the pre-treating and/or lysing agents prior to introducing thesample into a closed sterile system or to employ a specialized apparatuswhereby the treating agents may be contained within the closed systembut separate from the liquid filter medium until the apparatus is placedinto use. Apparatus of this type are disclosed in U.S. Pat. No.3,875,012, issued Apr. 1, 1975, and entitled "APPARATUS AND METHOD FORTHE DETECTION OF MICROBIAL PATHOGENS" and in U.S. Pat. No. 3,932,222,issued on Jan. 13, 1976 and entitled "FOR ISOLATING PATHOGENICMICROORGANISMS".

Furthermore when using the above-described improved method for detectingmicrobial pathogens in blood samples using a liquid filter medium in acentrifugation vessel which includes an injectable closure means at theend of the vessel against which the blood sample and liquid filtermedium are forced by centrifugation. It has been discovered that some ofthe heavier microbial pathogens which are received by the liquid filtermedium can pass, under the force imparted by centrifugation, completelythrough the liquid filter medium and come to rest adjacent the bottom ofthe centrifugation vessel being employed, and unless great care is takenduring separation and recovery of the liquid filter medium some of suchmicrobial pathogens can be left behind, unrecovered. It is believed thatthe loss of microbial pathogens, in such cases, occurs because, uponpassing completely through the liquid filter medium, the microbialpathogens become lodged in the tiny crevice formed between the wall ofthe centrifugation vessel and the injectable closure means.

Thus, it is desirable to have a closed, sterile method for separatingand concentrating microbial pathogens suspected to be present in a bloodsample without the necessity of having to premix the blood sample in apotentially contaminating environment and without the necessity ofemploying specially designed apparatus for accomplishing the pretreatingstep of the procedure. Furthermore, it is especially desirable toincrease recovery of the microbial pathogens which have been selectivelyseparated and concentrated from a sample fluid.

SUMMARY OF THE INVENTION

According to one embodiment of the present invention, a method ofdetection of microbial pathogens suspected to be contained in a samplefluid is provided. The procedure can be utilized on all types of bodyfluids, such as blood, bone marrow, spinal and pleural fluids, urine,and the like. In addition, the procedure can be utilized on any samplecontaining microorganisms to concentrate and separate the microorganismsfrom any antimicrobial factors present in the sample fluid, for example,foodstuff, such as milk, and the like. Generally, when employed inconnection with a blood sample the method includes depositing a lysedblood sample on a relatively thin layer of a high density, non-toxic,water immiscible, hydrophobic, liquid cushioning agent. Such cushioningagent is compatible with lysing and other blood heating agents and suchagents will rapidly separate from said cushioning agent when admixedtherewith. Therefore, preferably, to avoid possible contamination, ablood sample may be injected into a sterile confined zone containingboth the cushioning agent described above and an effective amount oflysing and/or other blood treating agents so that the blood sample istreated in situ. The lysed blood sample contained within the confinedsterile zone is then subjected to centrifugation which forces the bloodsample against the cushioning agent also contained therein. It has beendiscovered that upon such centrifugation, substantially all themicrobial pathogens contained in a lysed blood sample will pass out ofsuspension and collect in a layer adjacent the interface of thecushioning agent and the blood sample itself. The microbial pathogenswill actually penetrate the interface between the cushioning agent andblood sample and enter the cushioning agent or will remain on thesurface or adjacent the surface of the cushioning agent and none orsubstantially none will pass completely through the cushioning agent.This is to be contrasted with the use of a liquid filter medium whichselectively receives microbial pathogens and through which some of themicroorganisms may pass completely and become entrapped between the wallof the centrifugation vessel and injectable closure means. The layer ofmicrobial pathogens which so forms in accordance with the subjectinvention can then be recovered without substantial loss by separatingthe cushioning agent and a minor portion of the residual blood sampleadjacent the interface with the cushioning agent from the major portionof the sample. Thus, the presence of the cushioning agent duringcentrifugation of the lysed blood sample allows microbial pathogens tobe separated from the lysed blood sample as well as from any medicamentsand antipathogenic factors contained therein. Upon recovery, themicrobial pathogens can be analyzed both quantitatively andqualitatively.

Thus, in accordance with the present invention a high density,non-toxic, water immiscible, hydrophobic liquid cushioning agent for thecollection and separation of microbial pathogens from a sample fluid ina centrifugation zone is provided. I have discovered that the use ofsuch a cushioning agent either alone or in conjunction with a liquidfilter medium provides for a substantial increase in the recovery ofmicrobial pathogens which have been separated from a blood sample.Generally, inert liquids which are non-toxic to microbial organisms andwhich are water immiscible, hydrophobic and of high density can be usedas cushioning agents. The term "high density" as applied herein refersto a liquid which is of a density sufficient to not be supported by amixture of a blood sample and treating fluid therefor, or any otherfluid sample suspected of containing microbial pathogens in the presenceof centrifugal force. It is believed that these cushioning agentsbecause of their high density provide a cushioning effect to microbialpathogens which are forced to pass out of suspension in a blood sampleupon centrifugation. The microbial pathogens are caught, and cushionedadjacent the interface between the blood sample itself and thecushioning agent. In this manner, microbial pathogens are not lost tointerstitial spaces which may be present on the surface of the confinedsterile zone against which they have been forced by centrifugation.

In a further embodiment of the present invention, a novel article isprovided for recovering microbial pathogens from a sample fluid. Thenovel article comprises a smooth continuous surface within acentrifugation receptacle such that on centrifugation of thecentrifugation receptacle the sample fluid being centrifuged will beforced against the surface at a substantially perpendicular angle. Thesurface comprises the inner end of an injectable closure means for thereceptacle, which has said above described cushioning agent positionedthereon. Said cushioning agent will become distributed on the innersurface of said injectable closure means and will fill the interstitialspace between the wall of the centrifugation vessel and the injectableclosure means to prevent entrapment of any of the heavier microbialpathogens that may pass to said cushioning agent during centrifugation.When a swinging bucket type centrifuge is utilized, the inner surface ofsaid injectable closure means should be perpendicular to the bottom ofsaid injectable closure means. Furthermore, when an angle rotor typecentrifuge is employed, the smooth inner surface of the injectableclosure means is positioned within the centrifugation receptacle at anangle which is the complement of the angle at which centrifugation is tobe performed. This unique design enables one to use the commonlyavailable angle centrifuges and actually results in a shorter pathlength of travel for the pathogen through the sample fluid before itbecomes concentrated adjacent the inner face of the cushioning agent.This shorter path length results in a greater average force ("g" force)to be applied on the microbial pathogens within the sample fluid.Centrifugation with this unique designed centrifugation vessel willresult in the pathogens being concentrated adjacent the interface of thecushioning agent and the sample fluid and partially on the sidewall ofthe centrifugation container. The microbial pathogens thus concentratedare easily collected and removed from the centrifugation vessel. Thus,the positioning of the surface within the centrifugation receptacle atan angle such that the blood sample and cushioning agent will be forcedagainst the surface at a substantially perpendicular angle duringcentrifugation, insures that the cushioning agent will be disposedsubstantially uniformly across the surface so as to completely seal anyinterstitial spaces which will trap microbial pathogens. Sucharrangement provides a shorter path length of travel for pathogens andtherefore a greater average "g" force to be imparted upon pathogens toprovide a more efficient concentrated deposit thereof which can beeasily removed from the centrifugation vessel. Furthermore, the use ofthis specially designed centrifugation vessel results in an increasecentrifugation efficiency in that centrifugation can be carried out atless g's within the same time period as in conventional centrifugationor at the same g's in a shorter time period than the conventionalprocedure.

Furthermore, in accordance with a preferred embodiment of the subjectinvention, blood lysing and other blood treating agents which areemployed to prepare a blood sample can be positioned in an aqueoussolution within the centrifugation vessel in contact with the liquidcushioning agent. The liquid cushioning agent is of a higher densitythan said aqueous solution, and is hydrophobic and water immiscible andwill thereby support such aqueous solution. Furthermore, if thecentrifugation vessel becomes agitated during storage and shipment, thelysing or other blood treating agents will admix with the cushioningagent. However, once the centrifugation vessel is subjected tocentrifugation the high density liquid cushioning agent readilyseparates (sediments) and allows the blood treating agents to admix withthe blood sample within the vessel. This is a great advantage over theabove-described conventional liquid filter media which have ingredientswhich are incompatible with some of the blood treating agents andfurthermore since the conventional liquid filter media are aqueousbased, they will admix with the aqueous based blood treating agents ifthe two are in contact and prevent them from effectively diffusingtherefrom and treating a blood sample which is positioned within thevessel.

DETAILED DESCRIPTION OF THE INVENTION

This invention can be more easily understood from a study of thedrawings in which:

FIG. 1 is a cross sectional view of the preferred centrifugation articleof the present invention; and

FIG. 2-9 depict the steps of the improved method for detection ofmicrobial pathogens employing the article of FIG. 1.

Now referring to FIG. 1, a preferred embodiment of the improvedcentrifugation article of the present invention will be described. Asshown, the article 20 comprises an elongated, tubular centrifugationvessel 22 having a conventional injectable closure member 24 whichsealably closes the upper end thereof, and a novel injectable closuremember 26 which sealably closes the lower end thereof. When article 20is to be employed in the preferred embodiment of the method of detectingmicrobial pathogens of the present invention, an effective amount ofcushioning agent 28 and blood treating agents 30 can be depositedtherein.

Centrifugation vessel 22 can be made of siliconized glass or hardplastic, such as polycarbonate or polypropylene. Injectable closuremembers 24 and 26 can comprise rubber self-sealing stoppers. Injectableclosure members 24 and 26 both carry indentations 24a and 26a,respectively, to enhance the ease of injection by common types ofmedical injection needles. Evacuated space 32 is maintained at a lowerthan atmospheric pressure at a predetermined value so that thecentrifugation vessel can receive a known amount of liquid by injectionthrough injectable closure member 24 without excessive pressure beingbuilt up within the interior thereof which would cause injectableclosure members 24 and 26 to become dislodged from the openings withincentrifugation vessel 22.

Referring especially to injectable closure member 26 at the lower end ofcentrifugation vessel 22, it is noted that inner surface 34 ofinjectable closure member 26 is positioned at an angle with respect tothe walls of centrifugation vessel 22.

It is noted that article 20 is especially designed to be utilized withinan angle rotor centrifuge and that the angle of inner surface 34 is acomplement of the angle of the rotor. It should be noted, however, thatthe device of the subject invention can be utilized in a conventionalswinging bucket type centrifuge. In the latter instance, surface 34should be perpendicular to the bottom of the article 20 and is otherwiseutilized in the same general manner as will be described hereinbelow forthe article 20 illustrated in FIG. 1. Surface 34 should be smooth andsubstantially free of interstitial spaces and crevices in whichmicrobial pathogens could be entrapped. Further, the circular sealingarea around surface 34 where the material of injectable closure member26 meets the walls of the centrifugation vessel 22, should be tightlysealed so that the interface does not provide a large circular crevicein which microbial pathogens could become lodged.

The angle of incline of smooth surface 34 with respect to the walls ofcentrifugation vessel 22 is determined according to the centrifugationapparatus in which article 20 is to be centrifuged.

As discussed above, when a swinging bucket type centrifuge is utilized,surface 34 will be positioned perpendicular to the bottom of the article20. However, when an angle rotor centrifuge is utilized, surface 34 willcarry the complement of the angle of the rotor. Therefore, in generalwhen the rotor angles range from about 60 degrees to 10 degrees, theangle of surface 34, or angle of incline 36 within the centrifugationvessel will range correspondingly from 30 degrees to 80 degrees. Thus,the angle of incline, depicted by arc 36, will generally be thecomplement of the angle at which device 20 rests within the centrifugeduring centrifugation. For example, the angle of incline 36 depicted inFIG. 1 is approximately 34 degrees. Thus, for example, when article 20is placed in an angle rotor centrifuge in which centrifugation occurs atapproximately 56 degrees, fluids contained within article 20 will beforced against surface 34 at a substantially perpendicular angle.

It should be noted that as depicted in FIG. 1, injectable closure member26 is formed from a single piece of rubber. However, surface 34 can beprovided by employing a plug of material adjacent to the inner surfaceof a common rubber stopper such as injectable closure member 24, forexample. Such a plug can be manufactured from any of a number ofmaterials which provide a smooth surface, a good seal with the wall ofcentrifugation vessel 22, are injectable and which are non-toxic tomicrobial pathogens. A method of fabricating such a plug is to do so insitu by employing a material which can be poured into centrifugationvessel 22 once a common rubber stopper, such as injectable closuremember 24, has been placed in the lower end of centrifugation vessel 22.The material should be fluid enough and have setting times long enoughto allow centrifugation vessel 22 to be placed at the desired angle ofincline with the result that the material flows to the desired angle ofincline and then sets. Upon setting, the material will provide a smoothsurface 34 and a good seal with the walls of centrifugation vessel 22.Examples of such materials are common bathtub caulks and silicone baseresins which are provided in a low viscosity liquid from and which cureto form an elastomeric material. An example of the latter type ofmaterial is a silicone based fluid resin sold under the trade nameSYLGARD 134 by Dow Corning, Midland, Mich. When a material such asSYLGARD is employed, it is sometimes advisable to use a primer on theinterior wall of centrifugation vessel 22 in order to insure a good sealbetween the cured SYLGARD and the centrifugation vessel wall 22. Asuitable primer is sold under the trade name DC 1200 by Dow Corning.Thus, for example, smooth inclined surface 34 which is depicted in FIG.1 as the inner surface of a unitary injectable closure member 26 can beprepared by priming the interior wall of centrifugation vessel 22 with asuitable silicone base resin primer such as DC 1200, inserting a commonrubber stopper such as that depicted as injectable closure member 24,pouring an amount of a liquid silicone base resin such as SYLGARD 134into centrifugation vessel 22, placing the entire vessel at the desiredangle of incline and curing the silicone resin under appropriateconditions to form an elastomeric plug having a smooth surface 34positioned at the desired angle of incline adjacent a common rubberstopper. Bathtub caulking and similar materials may be employed in thesame general manner, if desired, and the correct angle of incline may beformed by centrifuging the article containing the uncured plug formingmaterial in the type of centrifuge with which the article is to beemployed.

Once smooth surface 34 has been formed either by placement of a unitaryinjectable closure member 26 or by a combination of a rubber stopper anda plug of material as described above, as effective amount of thecushioning agent of the subject invention can be added to the article20. The amount of cushioning agent employed should be sufficient tocompletely cover surface 34 upon centrifugation but not so large as tosignificantly limit the amount of blood which article 20 can receive.The amount of cushioning agent utilized can vary with the parameter ofthe particular system chosen, for example the stoppers design, volume ofthe residual blood and volatility of the cushioning agent utilized. Apreferred amount of cushioning agent can comprise from about 3.3% toabout 30.0% by volume based on the volume of the cushioningagent-residual blood sample mixture which is removed from article 20 andtested for the presence of microbial pathogens.

Generally, the cushioning agent of the subject invention can comprise ahigh density, hydrophobic, water immiscible liquid. As noted previously,the term "high density" as used herein refers to a liquid which wll notbe supported by the mixture of blood and blood treating fluid or anyother sample fluid suspected of containing microbial pathogens in thepresence of centrifugal force. In addition, the cushioning agent shouldbe non-toxic to microbial pathogens and relatively inert with respect tobutyl rubber, silicone rubber and other types of elastomers employed inthe manufacture of the injectable closure members described above. Thedensity of the cushioning agent can be in the range of from at leastabout 1.2 grams per cubic centimeter to about 2.0 grams per cubiccentimeter with the preferred range being from about 1.6 grams per cubiccentimeter to about 2.0 grams per cubic centimeter. Generally,fluorinated hydrocarbons having the above described characteristics andhaving molecular weights in the range of from about 300 to about 850 arepreferred. Furthermore, fluorinated hydrocarbons having the abovequalities which will evaporate at approximately the same rate as wateronce a sample containing the cushioning agent is placed on a common typeof culture plate are preferred. Therefore, cushioning agents having theabove described qualities and boiling points of at least about 200degrees F. to about 420 degrees F. and preferably of about 225 degreesF. to about 280 degrees F. can be utilized. The cushioning agent shouldalso have a vapor pressure which will not disrupt the injectable closuremeans from the tube during manufacturing steps such as autoclaving, forexample. Fluorinated hydrocarbons sold under the trade name FLUORINERTby 3M Company of Minneapolis, Minn., have been found to perform well ascushioning agents. Specifically, types FC-75, FC-48, and FC-43 of theFLUORINERT series have been found to be especially useful.

Although the exact function which such cushioning agents perform is notfully known, it is believed that they improve collection of microbialpathogens which have passed from suspension in a centrifuged bloodsample in at least two ways. First, the cushioning agent serves to sealinterstitial spaces, cracks, and crevices both on the smooth surface 34of the centrifugation vessel 22 and at the interface between the wallsof the centrifugation vessel 22 and injectable closure member 26. Thus,microbial pathogens which might otherwise become entrapped in suchinterstitial spaces, and therefore not recovered, are recovered with thecushioning agent 28 when it is removed from article 20. Secondly, it isbelieved that the cushioning agent does act to cushion the impact ofmicrobial pathogens which are forced out of suspension in a blood sampleduring centrifugation. This cushioning effect reduces the danger ofinjury to microbial pathogens which might otherwise occur upon impact.Further, while some of the microbial pathogens may actually pass intothe cushioning agent substantially none will pass completely through itand a majority will form on its surface at the interface between thecushioning agent 28 and the blood sample and collect in a layer.

After the cushioning agent 28 has been deposited within centrifugationarticle 20, water soluble treating agents 30 for the blood may also bedeposited therein. Such treating agents can include, for example, lysingagents and/or anticoagulants. Any suitable lysing agent can be utilizedso long as it is non-toxic to microorganisms. A suitable such lysingagent is an aqueous solution of a non-toxic saponin. It must be notedthat many saponins are known to be toxic to microbial pathogens.However, as set forth in Applicant's patent U.S. Pat. No. 3,883,425,issued May 13, 1975, and entitled "DETOXIFICATION OF SAPONINS", which isherein incorporated by reference, a method is disclosed for removing thetoxic ingredients from the heretofore thought to be toxic saponins. Ingeneral, the toxic saponin material can be detoxified in accordance withthe invention set forth in that patent and the resulting purifiedmaterial used within the scope of this invention. In addition, theaqueous solution of saponin can contain an anticoagulant and/or oxygenscavenger. A preferred anticoagulant is sodium polyanethol sulfonate(SPS) or heparin, for example. Sodium polyanethol sulfonate is preferredbecause it not only acts as an anticoagulant but also inhibits thephagocytic activity of granulocytes and monocytes and the normalantibacterial activity of blood serum. It is preferred that said aqueoussolution of treating agent be at least 1.0% by volume of the totalliquid in centrifugation vessel 22 including the treating solution,sample fluid and cushioning agent and preferably from about 7.6% toabout 17.5% by volume thereof.

Once the treating agents 30 have been deposited in centrifugationarticle 20, injectable closure member 24 can be put in place and space32 evacuated to the desired lower than atmospheric pressure.

Now referring to FIGS. 2-9, an analysis sequence is schematicallydepicted illustrating a preferred embodiment of the subject invention.As an example, a procedure which is carried out in accordance with oneembodiment of this invention for detection of microbial pathogens withina blood sample can be carried out conveniently with the followingapparatus:

The above described centrifugation article 20 containing the cushioningagent 28 and blood treating agents 30--the vessel can be of 12-14milliliters in volume.

A sterile glass syringe and one 11/2 inch 18 gauge disposable hypodermicneedle;

One sterile glass syringe and one 1 inch 21 gauge disposable hypodermicneedle;

One 5/8 inch 25 gauge hypodermic needle with cotton inserted at its hub(used as a vent);

Three blood agar plates;

One chocolate agar plate;

One Sabouraud plate.

It is noted that with the exception of centrifugation article 20 or someequivalent article, various types of well-known laboratory apparatus andculture media can be used to carry out the novel process of the subjectinvention. It is particularly noted that the culture media set forthabove are exemplary only and are generally preferred to be utilized fordetecting the most commonly known microbial pathogens. The blood agarplates suggested are conventionally utilized blood agar plates which arebasically sheeps blood and a base nutritional agent such as sugar, whichis held together with an agar solidifying agent on a Petri plate. Thechocolate agar plate is designed to grow certain fastidious pathogens,e.g., hemophilus. The Sabouraud plate is specifically designed to growfungi.

Thus, while various apparatus can be utilized in the method of thesubject invention, the above list of apparatus and materials can beconveniently utilized in the scope of this invention in a manner setforth below.

To utilize centrifugation article 20 set forth in FIG. 1 in the drawing,it is initially positioned so that injectable closure member 26 with itssmooth angled surface 34 is at the lower end of article 20 so that thecushioning agent 28 and blood treating fluid 30 rest upon smooth angledsurface 34. It should be noted that for purposes of illustration twoseparate liquid levels are depicted in FIG. 2 representing thecushioning agent 28 and treating fluid 30. In practice, a non stableemulsion or mixture of these two fluids may occur due to handling sothat two distinct liquid layers may not always be present. This unstablemixture of cushioning agent 28 and blood treating fluid 30 in no wayadversely affects the method set forth herein since separation of thetwo fluids rapidly occurs upon centrifugation.

Next, a predetermined amount of a blood sample 38 drawn from thepatient, for example 7 milliliters of blood, is injected into theevacuated space of centrifugation article 20 as depicted in FIG. 3 usinga common type of syringe 40. Alternately, the sample can be drawndirectly into article 20 using a standard and double needle fixturesupplied with conventional vacuum blood drawing devices such as soldunder the mark "Vacutainer" by Beckten Dickenson. Then, article 20containing the blood sample 38, the blood treating fluid 30, and thecushioning agent 28 is subjected to mixing to insure that the bloodtreating agents 30 are completely admixed with the blood sample 38. Thismixing step is depicted schematically by FIG. 4.

After the blood sample 38 has been treated in this manner,centrifugation article 20 is centrifuged to cause the microbialpathogens within the treated blood sample 42 to pass out of suspensionand collect adjacent the interface of the high density cushioning agent28 and the residual of the sample fluid. Some microbial pathogens willactually be deposited upon the sidewall of centrifugation vessel 22adjacent the high end of smooth surface 34 at point 22a. Thiscentrifugation step is represented schematically by FIG. 5. The speedand time of centrifugation can vary widely depending upon theconstruction material of centrifugation article 20 and the type ofcentrifugation apparatus. The centrifugation can be convenientlyaccomplished by imparting from between about 1500 and 6000 gravities andpreferably from about 1500 to 3000 gravities to the centrifugationarticle 20 containing the treated blood sample 42 and cushioning agent28. As depicted in FIG. 5, an angle rotor centrifuge is employed whichplaces the centrifugation article 20 at an angle of 56 degrees forexample, (depicted by arc 44) during centrifugation. Thus, if smoothangled surface 34 is at a 34 degree angle with respect to the interiorwalls of centrifugation article 20, the treated blood sample 42 andcushioning agent 28 will be forced against smooth angle surface 34 at arelatively perpendicular angle during centrifugation. It is noted thatwhen a swinging bucket type of centrifuge is employed, centrifugationarticle 20 will be centrifuged at substantially 0 degrees with respectto a horizontal surface. Thus, in such a case the angle of surface 34will be approximately 90 degrees and an injectable rubber closure memberhaving a flat inner surface can be substituted for injectable closuremember 26.

Once the centrifugation step has been completed, centrifugation article20 can be removed from the centrifuge and the major portion of thetreated blood sample 42 from which microbial pathogens have beenseparated can be removed. It is noted that, as used herein, the term"residual treated blood" or "residual blood" refers to a blood samplewhich has been centrifuged such that the microbial pathogens presenttherein have collected at the bottom of the sample, hence, leaving the"residual" portion of the sample substantially free of microbialpathogens. This step is depicted in FIG. 6. To aid in ease of removal, avent needle 44 in the form of a common hypodermic needle with cotton inits hub, for example, is injected through injectable closure member 24.A second hypodermic needle with syringe 45 attached can then be injectedthrough injectable closure member 26 to remove a major portion of theresidual treated blood sample 42 from which microbial pathogens havebeen separated. For example, when the centrifugation vessel has a volumeof from 12 to about 14 milliliters, a 11/2 inch 18 gauge needle can beemployed to remove all but about 1.3 to 1.7 milliliters of the treatedblood sample 42. As shown, it is preferred that the major portionresidual blood sample be drawn from the interior of centrifugationvessel 22 at a point opposite the sidewall adjacent the upper bevel endof smooth surface 34 to avoid disturbing the layer of microbialpathogens which has formed on and within the interface of the twoliquids and on the sidewall of centrifugation vessel 22 adjacent theupper end of said beveled smooth surface 34. The majority of theresidual blood is removed in this step however, a small portion of theresidual blood should be left in the centrifugation vessel 22 such thatof the total fluid remaining, the cushioning agent comprises from about4.7% to about 28.8% by volume. It is preferred that no more than about20% by volume shall be said cushioning agent because greater quantitiesof said cushioning agent may deleteriously effect the morphology ofmicrobial pathogen colonies in subsequent pathogen growth steps used inthe process.

Once the major portion of the treated residual blood sample has beenremoved, both needles may be withdrawn from injectable closure members24 and 26, and centrifugation article 20 is then subjected to a secondmixing step depicted schematically by FIG. 7. However, if desired, ventneedle 44 can be left in its position through injectable closure member24 to assist in removal of the pathogen containing fluid in a laterstep. The second mixing step serves to resuspend microbial pathogenswhich have separated from the major portion of residual treated bloodsample 42 and which have formed the layer described above. Resuspensionof the microbial pathogens so collected in the remaining minor portionof the residual treated blood sample 42 insures greater and more uniformrecovery.

Once the mixing step has resuspended the microbial pathogens in a minorportion of the residual treated blood sample 42, the mixture ofmicrobial pathogens in the residual treated blood sample and the highdensity cushioning agent can be removed from centrifugation article 20.This step is depicted in FIG. 8. As noted above, if desired, the ventinghypodermic needle 44 may be inserted through injectable closure member24 to allow easier removal of the remaining constituents. The syringe 46with attached hypodermic needle can then be injected through injectableclosure member 26 to draw out the mixture 48 of cushioning agent 28,minor remaining portion of residual blood sample 42 and microbialpathogens present therein. It is noted that particularly good recoverycan be obtained if the hypodermic needle used to remove theseconstituents is injected at the lower end of the angled smooth surface34. It is believed that the angle of surface 34 acts, in part, as afunnel into which the remaining fluid containing the microbial pathogensflow. The mixture 48 of high density liquid cushioning agent 28, and theremaining minor portion of the residual treated blood sample 42 with therecovered microbial pathogens should be approximately 11/2 millilitersof fluid. This fluid is then distributed on bacterial growth media. Thisstep is schematically illustrated in FIG. 9 in the drawing. With theapparatus set forth above, the material can be distributed as follows:

One blood agar plate can receive 0.3 milliliters of the mixture and theplate can be incubated at 36 degrees C. in an aerobic atmosphere. Twoblood agar plates can receive 0.3 milliliters of the aqueous solutionand can be incubated at 36 degrees C. in an anaerobic environment. Onechocolate agar plate can receive 0.3 milliliters of the aqueous solutionand can be incubated at 36 degrees C. in a candle jar. The Sabouraudplate can receive 0.3 millilters of the mixture and can be incubated at25 degrees C. in an aerobic environment. The growth media should bechecked daily for the presence of colonies. Microscopic analysistechniques can be employed. The number of microbial pathogens in onemilliliter of the blood can be determined by multiplying the number ofcolonies by a correction factor. This correction factor takes intoconsideration the recovery rate for a given organism, the volumes ofblood and high density cushioning agent employed and the amount of finalmixture plated. In the general example set forth above, the correctionfactor is 1.4.

It should again be noted that the exact procedural steps, apparatus andequipment, and types of culture media utilized in the detailedembodiment set forth above, can vary as desired. For example, any knownmeans can be utilized to admix the blood sample with the anticoagulantand/or lysing agent. Furthermore, in some cases, the step of withdrawalof the major portion of the blood sample 42 depicted in FIG. 6 can beeliminated completely and only a minor portion of the blood sample 42containing resuspended microbial pathogens can be withdrawn from thebottom of the centrifugation article 20 along with the high densitycushioning agent 28 as depicted in FIG. 8. Various other modificationscan be used in the procedure as desired.

EXAMPLE

The following example is given to better facilitate the understanding ofthis invention and is not intended to limit the scope thereof.

This example was performed in order to establish comparative dataregarding the percent recovery of microbial pathogens from blood sampleswhen the liquid filter medium technique set forth in Applicant'sprevious patent, U.S. Pat. No. 3,928,139, entitled "PROTECTION OFMICROBIAL PATHOGENS" issued Dec. 23, 1975, was employed as compared torecoveries of microbial pathogens obtained by employing the cushioningagent technique of the present invention.

The techniques employed in this example are identified in Table I aseither "Liquid Filter Medium" or "Cushioning Agent". The informationappearing in parenthesis directly beneath the technique identificationsets forth the liquid filter medium employed (e.g., 50% Sucrose) in theliquid filter medium tests and the cushioning agent employed (e.g.,Fluorinert FC-48) in the cushioning agent tests. Each sample tested(1-21 as listed in Table I) was prepared from 7 milliliter samples ofsterile lysed blood from healthy blood donors each sample beinginoculated with 0.3 milliliters of various known concentrations of ahuman pathogen (either Escherichia coli or Candida albicans asdesignated in Table I).

In each of the sample tests performed, a centrifugation article 20 asshown and described above was employed whether the liquid filter mediumtechnique or the cushioning agent technique was being used. In order toinvestigate the effect of employing a surface disposed at substantiallythe complement of the angle at which article 20 is centrifuged, some ofthe article 20 employed rubber stoppers having flat, smooth innersurfaces so that the inner surface of the bottom stopper was horizontalwith ground level when article 20 was standing upright. These bottomstoppers are identified as "Horizontal" in Table I. Also employed werespecially fabricated bottom closure members prepared by using commonrubber stoppers in conjunction with either common bathtub caulk or asilicone based resin sold under the trade name of SYLGARD 134 by DowCorning, Midland, Mich., to thereby prepare a bottom surface withinarticle 20 which, with respect to the walls of article 20, would have anangle substantially equal to the complement of the angle at whicharticle 20 was centrifuged. Samples tested using centrifugation articlesemploying the angled bottom stopper are identified as such in Table Iand the material employed (either bathtub caulk or SYLGARD) to preparethe angled surface is also set forth. The centrifugations performed inconjunction with each of the samples listed in Table I employing theangled bottom stopper were performed in an angle rotor centrifugewherein the angle of centrifugation was approximately 56 degrees. Thus,those centrifugation articles comprising angled bottom stoppers (angledat approximately a 34 degree angle) presented a bottom surface at asubstantially perpendicular angle to the direction of the centrifugalforce. Further, those samples employing a horizontal bottom stopper (asidentified in Table I) were centrifuged in a swinging-bucket typecentrifuge, which during operation causes centrifugation article 20 tobe spun in a plane substantially parallel with that of the ground. Thus,centrifugal force was exerted in a direction substantially perpendicularto the surface of the horizontal bottom stoppers.

When the liquid filter medium technique was employed, each of thearticles 20 contained 1.2 milliliters of an aqueous solution containing1.5 weight percent gelatin and the concentration by weight of sucroseindicated in Table I. Each of the articles 20 was inverted and chilledto 4 degrees C in an inverted position before the inoculated bloodsample was added. After each of the articles 20 had received therequisite amount of the blood sample containing the known amount ofhuman pathogen, it was placed within a water bath while still inverted.The water bath was set at 42 degrees C and the gelatin was allowed tomelt. Each tube was then centrifuged at approximately a 56 degree anglein a rotor centrifuge. Centrifugation was for a 30 minute period and wascarried out at a relative centrifugal force of 1500 or 3000 gravities asindicated in Table I.

At this point in the procedure used in conjunction with some of thesamples (hereinafter described as the "3" entry procedure), a 10milliliter syringe was inserted through the bottom stopper to removeabout 6.7 milliliters of residual blood sample. Then, centrifugationarticle 20 was subjected to mixing in a vortex mixer for about 1/2 to 2minutes. After the mixing step, 1.5 milliliters of the admixed filtermedium and that portion of the blood sample remaining in article 20 wereremoved by a common syringe with attached needle. In other samples, thestep of removing a major portion of the residual blood sample aftercentrifugation was not employed. In this case 1.5 milliliters of theliquid filter medium and blood sample adjacent the bottom stopper werewithdrawn from the bottom of centrifugation article 20 directly aftercentrifugation. The 1.5 milliliters, comprised of a mixture of liquidfilter medium and residual blood sample as well as the collectedmicrobial pathogens, was then admixed, after withdrawal from article 20,in order to provide a relatively even distribution of the componentsupon plating of the sample. The difference in these two procedures isindicated in Table I under the notation "Number of Entries" whichdesignates each test sample as following the procedure having either twoor three entries. In the case of three entries, the first entry is forinjection of the blood sample, the second entry is for the removal ofthe majority of the blood sample after centrifugation but before mixing,and the third entry is for removal of the remaining liquid filter mediumand the remaining portion of the blood sample. In the two entryprocedures, the first entry is for injection of the blood sample intoarticle 20 and the second entry is for withdrawal from the bottom of thecentrifugation article 20 of approximately 1.5 milliters of admixedliquid filter medium and blood sample.

In both cases, whether the two entry or three entry procedure wasemployed, five samples containing 0.3 milliliters each of the 1.5milliliters drawn from the bottom of article 20 was plated on 5 separateplates. After incubation, these samples were compared with controlplates prepared by adding the same known quantity of microbial pathogensas was injected into the tested blood sample to a saline solution andplating 0.3 milliliters of this saline solution on each of 5 agarplates. The percent recovery based on the comparison of the sampleplates to the control plates is listed in Table I.

The procedure used with regard to each sample of blood tested using thecushioning agent technique was as follows. A centrifugation article 20was prepared using either a horizontal or angled bottom stopper asdiscussed above. Each centrifugation article 20 used in the cushioningagent procedure contained 0.3 milliliters of Fluorinert FC-48. Sevenmilliliters of lysed blood which had been contaminated with a knownnumber of microbial pathogens was then injected through the top stopperof article 20. The next step was to centrifuge each of the articles 20in the same angle rotor centrifuge employed in the liquid filter mediumtechnique. Centrifugation time was 30 minutes and the relativecentrifugal force applied was either 1500 gravities or 3000 gravities asshown in Table I. When the 3 entry technique was employed, approximately5.8 milliliters of residual blood sample was withdrawn by injection of a10 milliliter syringe through the bottom stopper. When the two entrysystem was employed, no such entry or withdrawal was made. In eithercase 1.5 milliliters, comprising approximately 1.2 milliliters ofresidual blood sample and 0.3 milliliters Fluorinert was withdrawn fromthe bottom of centrifugation article 20. Five sample plates were thenprepared utilizing 0.3 milliliters of the admixed residual blood sampleand Fluorinert in each case. These sample plates were incubated andsubsequently compared with control plates which had been prepared in thesame manner as discussed above in connection with the liquid filtermedium technique. The percent recovery when employing the cushioningagent technique was then computed and is set forth in Table I.

While an exact theory to explain the data obtained pursuant to thisexperiment cannot be set forth, it is believed that the results obtainedsupport Applicant's theory with regard to the present invention. Forexample, the low recovery rate of Sample No. 2 of Table I (wherein theliquid filter medium technique was employed using 50% sucrose and ahorizontal bottom stopper) could be a result of microbial pathogensbecoming trapped along the interior edge of the horizontal bottomstopper next to the wall of centrifugation article 20. Comparing theresults of Sample No. 1 with those of Sample No. 3, which again employedthe liquid filter medium technique and a horizontal stopper but wasperformed using the three entry procedure, it is seen that the recoveryrate in Sample No. 3 is greatly improved. This could be explained by thefact that because less relative centrifugal force was applied, themicrobial pathogens did not become so tightly lodged in the crevicebetween the bottom stopper and the walls of article 20. Further, the 3entry technique allows for recovery of all microbial pathogens whichhave been resuspended in the remaining portion of the blood sample. Whenthe two entry procedure is employed some of the resuspended microbialpathogens may be left behind in the residual portion of the bloodsample.

A comparison of Sample No. 15 with that of Sample No. 17 demonstratesthe improved results possible when using the cushioning agent techniqueof the present invention. In Sample No. 15, as in Sample No. 17, ahorizontal bottom stopper was employed, the number of entries was 3, andthe relative centrifugal force was the same as in Sample No. 17 (3000).The improved results when using the cushioning agent technique isbelieved to result from the use of the cushioning agent which does notallow microbial pathogens to pass therethrough and become lost at theinterface between the walls of article 20 and the bottom stopper.

Further, a comparison of Sample No. 9 with Sample No. 11 demonstratesthe advantages obtained by employing an angle stopper as compared to thehorizontal bottom stopper. Since the conditions and techniques inSamples No. 9 and 11 are exactly identical except for the fact that inSample No. 11 an angle stopper was employed, the improvement present inrecovery rate in Sample No. 11 over that of Sample No. 9 can only beattributed to the use of an angled bottom stopper positioned at an anglewith respect to the walls of article 20 which is the complement of theangle at which article 20 was centrifuged. A comparison of Sample No. 10to Sample No. 12 similarly illustrates the superior results obtainedwhen using an angled bottom stopper at higher relative centrifugalforces (3000).

                                      TABLE I                                     __________________________________________________________________________                                    Number                                        Sample                                                                            Microbial          Bottom   of   RCF (Relative                                                                           %                              No. Pathogen                                                                              Technique  Stopper  Entries                                                                            Centrifugal Force)                                                                      Recovery                       __________________________________________________________________________     1. Escherichia coli                                                                      Liquid Filter Medium                                                                     Horizontal                                                                             2    1500      65 ± 11                                 (50% Sucrose)                                                      2. "       Liquid Filter Medium                                                                     Horizontal                                                                             2    3000      55 ± 5                                  (50% Sucrose)                                                      3. "       Liquid Filter Medium                                                                     Horizontal                                                                             3    1500      90 ± 4                                  (50% Sucrose)                                                      4. "       Liquid Filter Medium                                                                     Horizontal                                                                             2    3000      63 ± 14                                 (60% Sucrose)                                                      5. "       Liquid Filter Medium                                                                     Horizontal                                                                             3    1500      90 ± 3                                  (60% Sucrose)                                                      6. "       Liquid Filter Medium                                                                     Horizontal                                                                             3    3000      95 ± 15                                 (60% Sucrose)                                                      7. "       Cushioning Agent                                                                         Horizontal                                                                             2    1500      65 ± 16                                 (Fluorinert FC-48)                                                 8. "       Cushioning Agent                                                                         Horizontal                                                                             2    3000      90 ± 7                                  (Fluorinert FC-48)                                                 9. "       Cushioning Agent                                                                         Horizontal                                                                             3    1500      81.5 ± 4                                (Fluorinert FC-48)                                                10. "       Cushioning Agent                                                                         Horizontal                                                                             3    3000      89 ± 2                                  (Fluorinert FC-48)                                                11. "       Cushioning Agent                                                                         Angled   3    1500      98 ± 1.2                                (Fluorinert FC-48)                                                                       (Sylgard Plug)                                         12. "       Cushioning Agent                                                                         Angled   3    3000      98 ± 1                                  (Fluorinert FC-48)                                                                       (Sylgard Plug)                                         13. Candida albicans                                                                      Liquid Filter Medium                                                                     Horizontal                                                                             2    1500      38 ± 22                                 (50% Sucrose)                                                     14. "       Liquid Filter Medium                                                                     Horizontal                                                                             2    3000      40 ± 8                                  (50% Sucrose)                                                     15. "       Liquid Filter Medium                                                                     Horizontal                                                                             3    3000      53 ± 16                                 (50% Sucrose)                                                     16. "       Liquid Filter Medium                                                                     Horizontal                                                                             3    1500      92 ± 7                                  (60% Sucrose)                                                     17. "       Cushioning Agent                                                                         Horizontal                                                                             3    3000      98 ± 6                                  (Fluorinert FC-48)                                                18. "       Cushioning Agent                                                                         Angled   3    1500      99 ± 4                                  (Fluorinert FC-48)                                                                       (Sylgard Plug)                                         19. "       Cushioning Agent                                                                         Angled   3    3000       100                                       (Fluorinert FC-48)                                                                       (Sylgard Plug)                                         20. "       Cushioning Agent                                                                         Angeled (Bathtub                                                                       3    1500      91 ± 16                                 (Fluorinert FC-48)                                                                       Calk Plug)                                             21. "       Cushioning Agent                                                                         Angled (Bathtub                                                                        3    3000      99 ± 1                                  (Fluorinert FC-48)                                                                       Caulk Plug)                                            __________________________________________________________________________

While this invention has been described in relation to its preferredembodiments, it is to be understood that various modifications thereofwill now be apparent to one skilled in the art upon reading thisspecification and it is intended to cover such modifications as fallwithin the scope of the appended claims.

I claim:
 1. An article used for the concentration of microbial pathogensfrom a sample fluid comprising:an enclosed centrifugation receptaclesealably closed with injectable closure means, the interior of saidreceptacle comprising an evacuated spaced maintained at a lower thanatmospheric pressure, adjacent a high density, non-toxic to microbialorganisms, water immiscible, hydrophobic liquid cushioning agent capableof collecting substantially all of the microbial pathogens which passout of suspension from said sample fluid without a loss of saidmicrobial pathogens to interstitial spaces present in saidcentrifugation receptacle and wherein the density of the liquidcushioning agent is sufficient so as not to be supported by a mixture ofa sample fluid and a treating fluid therefore.
 2. The article of claim 1wherein said cushioning agent is present in an amount equal to fromabout 4.7% to about 28.6% by volume.
 3. The article of claim 2 whereinsaid cushioning agent comprises an inert, liquid fluorinated hydrocarbonhaving a density of from about 1.6 grams per cubic centimeter to about2.0 grams per cubic centimeter.
 4. The collecting agent of claim 3wherein said liquid fluorinated hydrocarbon has a boiling point of fromat least about 200 degrees F. to about 420 degrees F.
 5. An article forthe concentration of microbial pathogens contained in a blood samplecomprising:(a) an enclosed centrifugation receptacle having a first endand a second end, said ends being sealably closed with injectableclosure means; (b) an injectable plug adjacent the injectable closuremeans of said second end, said injectable plug comprising a surfacedisposed at an angle with respect to the interior of said centrifugationreceptacle, said angle being the complement of the angle at which saidarticle is to be centrifuged; (c) an effective amount of a high density,non-toxic to microbial organisms, water immiscible, hydrophobic liquidcushioning agent contained within said centrifugation receptacle andwherein the cushioning agent is capable of collecting substantially allof the microbial pathogens which pass out of the suspension from saidsample fluid without loss of said microbial pathogens to interstitialspaces present in said centrifugation receptacle and wherein the densityof the liquid cushioning agent is sufficient so as not to be supportedby a mixture of a sample fluid and a treating fluid therefore; and (d)an effective amount of a non-toxic lysing agent contained within saidcentrifugation receptacle.
 6. The article of claim 5, wherein saidinjectable plug is an extension of the injectable closure means of saidsecond end of said centrifugation receptacle.
 7. The article of claim 5wherein said cushioning agent is present in an amount equal to fromabout 3.3% to about 30.0% by volume.
 8. The article of claim 5 whereinthe angle of the surface of said injectable plug with respect to theinterior of said centrifugation receptacle is from about 30 degrees toabout 80 degrees.
 9. The article of claim 8 wherein said angle is 34degrees.
 10. The article of claim 5 wherein the angle of the surface ofsaid injectable plug with respect to the interior of said centrifugationreceptacle is about 90 degrees.
 11. The article of claim 5 wherein saidcushioning agent is an inert, liquid fluorinated hydrocarbon.
 12. Thecushioning agent of claim 11 wherein said fluorinated hydrocarbon has adensity of at least 1.2 grams per cubic centimeter and a boiling pointat least about 200 degrees F.
 13. In an article for the concentration ofmicrobial pathogens suspected to be present in a blood sample,comprising:an enclosed centrifugation vessel having a first end and asecond end; a chamber containing an evacuated space maintained at alower than atmospheric pressure; and a first injectable closure meanssealably closing the first end of said centrifugation vessel, theimprovement comprising: a second injectable closure means sealablyclosing said second end of said centrifugation vessel and comprising asurface disposed at an angle with respect to the interior of saidelongated centrifugation receptacle, said angle being the complement ofthe angle at which said article is to be centrifuged; and an effectiveamount of an inert, non-toxic to microbial organisms, high density,water immiscible, hydrophobic liquid cushioning agent contained withinsaid chamber and wherein the cushioning agent is capable of collectingsubstantially all of the microbial pathogens which pass out of thesuspension from said sample fluid without loss of said microbialpathogens to interstitial spaces present in said centrifugationreceptacle and wherein the density of the liquid cushioning agent issufficient so as not to be supported by a mixture of a sample fluid anda treating fluid therefore.
 14. The improved article of claim 13 andfurther comprising an effective amount of blood treating agents selectedfrom the group consistng of lysing agents, anticoagulants and mixturesthereof.
 15. The article of claim 14 wherein said treating agents arepresent in an amount equal to at least about 1% by volume based on thevolume of the treating agents, blood sample and cushioning agent. 16.The article of claim 15 wherein said treating agents are presnt in anamount equal to from about 7.6% to about 17.5% by volume.
 17. Thearticle of claim 14 wherein said treating agent comprises a detoxifiedsaponin.
 18. The article of claim 14 wherein said treating agentcomprises sodium polyanothal sulfonate.
 19. The article of claim 13wherein said angle of the surface of the injectable closure meanssealably closing said second end of said centrifugation vessel is fromabout 30 degrees to about 80 degrees.
 20. The article of claim 19wherein said angle is 34 degrees.
 21. The article of claim 13 whereinsaid cushioning agent is present in an amount equal to from about 3.3%to about 30.0% by volume.
 22. The article of claim 21 wherein saidcushioning agent comprises a liquid fluorinated hydrocarbon having adensity of from about 1.6 grams per cubic centimeter to about 2.0 gramsper cubic centimeter, and a boiling point of at least about 200 degreesF.